The present invention relates to a set of plant promoters which can be induced by biotic or abiotic stresses, in particular by pathogens, to their use, to expression vectors comprising said promoters and a gene of interest, and to cells and/or plants transformed with said vectors. The invention also relates to methods for obtaining said cells or plants, said transformed plants exhibiting improved resistances to said pathogens.
Diseases, whether they are of fungal, bacterial or viral origin, are the major problem in viticulture, both in terms of the quality of the musts and of the wines produced (for example Botrytis cinera, which is a grey mould agent which attacks the berries at grape-harvesting and leads to bad tastes in the wines), drops in production (for example foliar diseases such as grape downy mildew or grape powdery mildew, attacks by Botrytis on flowers or Fan leaf disease of the grapevine linked to the presence of the virus G.F.L.V., Grape Fan Leaf Virus), or even to that of the survival of the vineyard (for example wood diseases such as eutypiosis or esca syndrome). The conventional control arsenal ranges from simple prophylaxis to plant-protection treatments, biological control to date being used very little.
Chemical control is of course the most widely used method, even though treatments are increasingly being tempered (models for forecasting risks of disease for grape downy mildew for example). With regard to fungicides for example, the French vineyard, which represents approximately 10% of the cultivated land in France, each year uses close to 40% of the fungicides consumed in this country. On a European level, on close to 4 million hectares of vineyard, the 9 to 10 treatments which are carried out each year to control these diseases lead to the use of 120,000 tonnes of fungicidal products.
To cite just the problem of grey mould, it is estimated that over the years 1992 to 1993, 25 to 30% of the 3.7 million hectares of European vineyard were concerned, for a cost of plant-protecting products of 97 and 69 million German Marks (DM), respectively.
The use of these products is not without consequence for the environment (it is the case, for example, for the soil fumigants used to destroy nematodes, which are vectors of the grape fan leaf virus). It also sometimes poses technological problems, with difficulties which can occur during fermentations (the use of sterol biosynthesis inhibitors can block yeast growth at the end of fermentation) and commercial difficulties (procymidone, an anti-Botrytis product, which is sometimes found in wines, hence the American dispute in the years 1990 to 1991).
Moreover, the use of these products has already led to the appearance of resistant strains. This phenomenon has been particularly marked in Champagne and, for some years, the xe2x80x9cComitxc3xa9 Interprofessionnel des Vins de Champagnexe2x80x9d (CIVC) [Interprofessional Committee of the Wines of Champagne] has recommended not treating against Botrytis.
To overcome these drawbacks, it is imperative to balance the use of plant-protecting products by developing novel methods of control in order to considerably decrease the amounts of products spread over vineyards.
Two approaches are currently envisaged:
Reinforce prophylaxis and decrease the amount of products used (cultivation methods and preventive control, models for forecasting risks of disease, novel spreading materials, novel, more degradable molecules, etc.).
Improve the resistance of the grape varieties to disease.
For this second approach, conventional genetic improvement via the sexual pathway (hybridization with tolerant varieties) is impossible according to French legislation on Appellations d""Origine Contrxc3x4lxc3xa9e (A.O.C.) (registered designations of origin) which imposes the grapevine varieties which are to be used for a given appellation (designation). In addition, technically, grapevine, which is a ligneous plant, would require several tens of years to integrate one or more novel resistance properties while at the same time conserving the biochemical and aromatic properties of the grapevine varieties, which are factors of the organoleptic quality of the wines produced.
The control of the regeneration and of the genetic transformation of the grapevine, which has been carried out by the research team of the laboratories of the applicant company since 1988-1990, has made it possible to envisage using the modern techniques of cellular and molecular biology to increase the tolerance of grapevine varieties to fungal diseases.
Henceforth, it is possible, on the one hand, to integrate, into the genome of the grapevine, one or more homologous or heterologous genes which enable the overexpression or expression of a molecule of interest, which is of protein or other nature, and/or the opening of a new biosynthetic pathway and, on the other hand, to regenerate a plant which is more tolerant to one or more diseases, i.e. which has defense mechanisms which are reinforced with respect to the pathogen(s) in question.
There are several different mechanisms of this type in plants. Some can be regarded as being passive and are linked to the physicochemical properties of the cells, epidermal tissues and/or organs of the plant (for example the cuticle or the morphological properties of the grape cluster). Others belong to the dynamics of gene/gene interactions (plant resistance genes and pathogen avirulence genes, mechanisms of host/parasite interactions). These interactions can lead to the development of hypersensitivity reaction (rapid death of the cells of the plant around the point of infection in order to block the colonization of the plant by the fungus, bacterium or virus), but also to the synthesis and to the accumulation of a whole series of compounds. Among these, some can be parietal constituents which are involved in the formation of a xe2x80x9cphysicalxe2x80x9d barrier around the point of infection (callose, lignin, hydroxyprolinerich protein, etc.), and others are molecules having antimicrobial functions which are more or less well defined (phytoalexins, pathogen-associated proteins: PR proteins (pathogenesis-related protein), etc.).
The overexpression of these molecules which have antimicrobial functions or which are involved in the formation of a physical barrier around the point of infection can provide plants with a xe2x80x9cnaturalxe2x80x9d resistance in response to stresses, in particular stresses of microbial type.
However, constitutive overexpression of this type of protein cannot be envisaged without drawbacks for the plant (energetic cost, slowing down of growth, etc.), which is why it is necessary to envisage the use of inducible promoters and in particular of promoters which are inducible by the stress itself. This is precisely the subject of the present invention.
The present invention relates to a nucleic acid sequence chosen from the group comprising:
a) the IND S1 sequence,
b) any sequence corresponding to a fragment of the IND S1 sequence and having a promoter sequence effect in plants.
The invention also relates to promoters in plants, chosen from the group comprising:
a) the promoter PMs PR10-1 corresponding to the IND S1 sequence,
b) a promoter of PMs PR10-1 type corresponding to a sequence according to the invention.
Said promoters of PMs PR10-1 type are preferred which exhibit at least 80% homology with the IND S1 sequence. Those which exhibit at least 90% or 95% homology with said sequence are particularly preferred. The promoter sequences in plants which are characterized in that they comprise at least one sequence which is identical to those of the abovementioned promoters are also included in the present invention.
The subject of the invention relates most particularly to the use of the promoters according to the invention for the tissue-specific or non-tissue-specific expression of a gene in a way which is inducible in plants by a biotic or abiotic stress.
Among said biotic stresses according to the invention, the biotic stresses engendered by the attack of a parasite such as a virus, bacterium, yeast or fungus are particularly preferred.
Among said abiotic stresses according to the invention the abiotic stresses engendered by a mechanical wound, such as that caused in particular by an insect or by a physical phenomenon such as wind or frost, are particularly preferred.
The promoters or promoter sequences according to the invention can be used to prepare systems for expression in plants, the systems being able to be inducible and/or constitutive depending on the plant tissues or organs transformed (cf. Examples 2, 3 and 4).
These promoters were obtained from the regulatory sequences of PR protein genes in lucerne. Advantage was taken of the incompatibility response (hypersensitivity reaction, HR) obtained in the host/parasite relationship between lucerne (Medicago sativa) and Pseudomonas syringae pv pisi in order to study the promoter which is responsible for this reaction.
When Pseudomonas attacks lucerne, the appearance of a plant reaction is observed in the region of infection.
The plant material was thus removed following the bacterial attack in order to construct a cDNA library from the messenger RNAs produced in the infected regions adjacent to the necrosis. An amplification by polymerase chain reaction (PCR), using synthetic polynucleotides corresponding to motifs which are conserved in PR protein genes of leguminous plants, made it possible to obtain a radioactive probe which was then used to select transcripts in the cDNA library. Among these, one of them was retained since, after sequencing, it exhibited good homology with equivalent genes encoding PR proteins which are known for other plants (cf. FIGS. 1 and 1a which represent the general scheme of the method for isolating the promoter).
The analysis showed that it corresponded to a gene encoding a class 10 PR protein according to the van Loon (1994) classification. This gene was therefore designated Ms PR10-1 (Medicago sativa class 10 PR protein, clone 1).
A subject of the present invention is also systems which are for expressing a gene in plants and which are characterized in that they comprise at least the sequence of said gene under the control of a promoter or of a sequence according to the invention. Among the expression systems according to the invention, expression vectors, and in particular plasmid-type expression vectors, are preferred. Advantageously, said expression vectors are characterized in that they can be transferred into strains of Agrobacterium.
A subject of the invention is also a system or vector for expressing a gene in plants according to the invention, characterized in that it is inducible in plants by a biotic or abiotic stress, preferably a biotic or abiotic stress such as those described above.
The invention also relates to the systems or vectors according to the invention, characterized in that said gene is a gene of interest.
A gene is considered to be a gene of interest if its modification may be desired or used in any type of industry, including agriculture. Besides the already mentioned agriculture, industries such as, for example, the agrofoods industry, the cosmetics industry, the pharmaceutical industry, the chemical industry, etc. will come to mind. This list of examples is not, of course, limiting.
The gene may thus be, for example, a gene of agronomic interest or a gene which enables the plant to produce substances having a value for human or animal nutrition or health. Among the genes of agronomic interest, please note, for example, any gene whose expression makes it possible to modify the physiology of the plant, such as in particular inhibiting, slowing down, accelerating or triggering steps or phenomena which are involved at a given period in the life of the plant, or any gene whose expression makes it possible to improve or decrease the resistance of the plant to physical, chemical or biological attacks. Among the genes of interest which enable the plant to produce substances having a value for human or animal nutrition or health, is meant, for example, the genes which encode pharmaceutical or enzymatic compounds (which can be used for the biosynthesis or biodegradation of organic compounds) or compounds with nutrient value, or genes which make it possible to modify or to inhibit the expression of pharmaceutical, nutrient or toxic compounds or of aromas.
The invention comprises in particular the systems or vectors according to the invention, characterized in that said gene of interest is a gene which is involved in the response to a biotic or abiotic stress, preferably in the response to the inducing biotic or abiotic stress.
Preferably, the invention relates to the systems or vectors according to the invention, characterized in that the biotic stress is the attack of a parasite and the gene of interest is a gene of resistance to said parasite.
Even more preferably, the invention comprises the systems or vectors according to the invention, characterized in that the parasite is a virus, a bacterium, a yeast or a fungus, and the gene of interest is a gene which is involved in the synthesis of a molecule with anti-pathogen action, preferably a gene which is involved in the synthesis of phytoalexins or of PRs.
The constructs which enable the expression of these genes may of course comprise, besides the gene of interest, in particular coding strand 3xe2x80x2-end polyadenylation sequences, as well as enhancer sequences of said gene or of a different gene.
Of course, the constructs will have to be adapted in order to ensure that the gene will be read in correct reading frame with the promoter, and it will obviously be possible to envisage using if this is necessary, several promoters of the same type, as well as several enhancer sequences.
It is also possible to express, with the aid of the promoters according to the present invention, several genes, which are either placed in cascade or carried by different expression systems.
Among the genes of interest which can be expressed by the constructs according to the present invention, mention should be made of the genes which can be placed under the control of the promoter PMs PR10-1 in order to trigger mechanisms of resistance to the plant pathogens which are viroids, viruses, phytoplasmas, bacteria and fungi, or also even the resistance to insects or to ravages (the promoter also being inducible, in tobacco in particular, by wounds).
Among the possible strategies, reference may be made to the reviews by LAMB et al., 1992; VAN LOON et al., 1994; BROOGLIE and BROOGLIE, 1993; CHET, 1993 and to that by PAPPINEN et al., 1994.
By way of example, mention may be made, whether they are homologous or heterologous, of the genes encoding:
hydrolytic enzymes such as chitinases (BROOGLE et al., 1991) or xcex21-3 glucanases (KAUFFMAN et al., 1987), or combinations of genes encoding these two enzymes,
PR proteins (VAN LOON et al., 1994) such as osmotin (LIU et al., 1994, ZHU et al., 1995), thaumatin-like PR-proteins (VIGERS et al., 1992) or class 1 PR proteins (CUTT et al., 1989; HAHN K. and STRITTMATTER G., 1994),
RIP proteins (ribosome Inactivating Protein) of plants (LOGEMANN J. et al., 1992) or of other organisms or microorganisms,
proteins which have an inhibitory role for fungus attack enzymes: protease inhibitor (MASOUD et al., 1993), polygalacturonase-inhibiting proteins (TOUBART et al., 1992) or other inhibitory proteins,
lectins or chitin binding proteins (BROEKAERT et al., 1989; LERNER and RAIKHEL, 1992),
proteins such as (T4) phage or mammalian lysozyme (DURING et al., 1992),
proteins which are involved in the phytoalexin biosynthetic pathway (HAIN et al., 1993),
antimicrobial peptides such as defensin (BROEKAERT et al., 1995; VIGERS et al., 1991; TERRAS et al., 1995),
pathogen-resistance proteins (DE WIT, 1992) which are involved in or trigger hypersensitivity reactions (STRITTMATTER et al., 1995) or enzymes which lead to the production of hydrogen peroxide (WU et al., 1995),
proteins which lead to the production of antifungal, antibacterial or anti-insect toxins (KINAL et al., 1995),
proteins with peroxidase function which can intervene in lignin polymerization (LAGRIMINI et al., 1987) or in other oxidative reactions (BRADLEY et al., 1992),
proteins of viral origin, such as proteins of the shell of viruses in the sense or antisense position (see BEJARANO and LICHTENSTEIN, 1992),
proteins which are involved in the synthesis of molecules which trigger the signal transduction chain of stress mechanisms (jasmonic acid, salicylic acid, etc.) or proteins which are themselves involved as a stress signal or intermediates in the transduction chain (systemin, GTP binding proteins) for this, see SCHEEL et al., 1991, VERMA et al., 1994; VERA-ESTRELLA et al., 1994),
proteins which are toxic for insects (VAECK et al., 1987).
This list is not limiting.
A subject of the present invention is also plant cells which are transformed with a system or vector according to the present invention. Advantageously, said plant cells are grapevine cells and the gene of interest is a gene which confers resistance to a parasite.
The present invention also relates to methods for obtaining cells, characterized in that plant cells are transformed with the aid of a microbiological method which includes an expression system or a vector according to the invention.
Among the most widely used transformation methods, mention should be made in particular of the methods which use Agrobacterluin, whether it is Agrobacterium tumefaciens or Agrobacterium rhizogenes. 
These methods are known, and they will not be described again in detail.
This technology, using plasmid systems, makes it possible to carry out a first transformation of a strain of competent bacteria, generally E. coli, which makes it possible to control the structure of the plasmids, and then the strain is used to transfer the recombinant plasmids into strains of agrobacteria which will then be used to transform the plant cells.
The present invention also relates to the transformed plant cells obtained by this method.
The invention also comprises a method for obtaining a plant expressing a gene of interest, characterized in that plant cells of said plant are transformed with the aid of a system or of a vector according to the invention, the cells expressing the gene of interest are selected and a plant is regenerated from said selected cells.
The invention also comprises the plants which comprise a system or a vector according to the invention, and/or cells according to the invention, preferably the plants obtained by implementing a method according to the invention.
It should in particular be noted that the constructs according to the present invention and which use the inducible promoters have made it possible to transform diverse plants, in particular tobacco (Nicotiana benthamiana), lucerne (Lotus corniculatus) and also grapevine (Vitis sp.).
It has also been possible, moreover, to demonstrate the advantage of the promoter according to the present invention during the regeneration of plants from cells. Specifically, the promoters of equivalent constructs which use strong constitutive promoters have never made it possible to obtain regenerated plants and it might be that the production of defense proteins leads very rapidly to the necrosis of the cells, thus preventing regeneration. This is an additional advantage of the constructs according to the present invention in some systems of plant transformation and regeneration.
Other properties and advantages of the constructs and of the methods according to the present invention may be demonstrated in the examples which follow.